Apparatus and method for improved optical detection of particles in fluid

ABSTRACT

A number of fluidic-photonic devices for allowing optical detection, systems employing such devices, and related methods of operation and fabrication of such devices are disclosed herein. In at least some embodiments, the devices can serve as flow cytometry devices and/or employ microfluidic channels. Also, in at least some embodiments, the devices are fluidic-photonic integrated circuit (FPIC) devices that employ both fluidic channels and one or more waveguides capable of receiving and/or delivering light, and that can be fabricated using polymeric materials. The fluidic-photonic devices in at least some embodiments are capable of functionality such as on-chip excitation, time-of-flight measurement, and can experience enhanced fluorescence detection sensitivity. In at least some embodiments, the devices employ detection waveguides that are joined by way of a waveguide demultiplexer. In additional embodiments, a variety of techniques can be used to process information received via the waveguides, including an iterative cross-correlation process.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims priority toU.S. patent application Ser. No. 13/605,925, filed on Sep. 6, 2012,which is a divisional application of and claims priority to U.S. patentapplication Ser. No. 12/091,414, filed on Apr. 24, 2008, which is a U.S.National Stage application under 35 U.S.C. §371 of International PatentApplication No. PCT/US2006/060313, filed on Oct. 27, 2006, which claimsbenefit of priority of U.S. Provisional Patent Application No.60/731,551, filed on Oct. 28, 2005. The entire contents of thebefore-mentioned patent applications are incorporated by reference aspart of the disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support awarded bythe following agencies: Air Force Office of Scientific Research (AFOSR)Grant No. F49620-02-1-0288. The United States Government has certainrights in this invention.

FIELD OF THE INVENTION

The present invention relates to systems and methods for opticaldetection and, more particularly, to systems and methods for detectingsmall objects or particles such as cells or DNA.

BACKGROUND OF THE INVENTION

Over a period of nearly five decades, flow cytometry has evolved from asimple technique for counting suspended particles (e.g., analytes, cellsor DNA) in fluid into a highly sophisticated and versatile techniquethat is critical to clinical diagnosis and fundamental biomedicalresearch. Early efforts in the development of flow cytometry focusedupon the attainment of a stable flow system able to transport particles,without disturbance by any alien aerosol, to regions of laser beamillumination for optical interrogation via fluorescence or lightscattering. A standard approach for today's flow cytometers is to createa laminar sheath flow in a transparent capillary tube to minimize noisedue to fluctuations in position and propagation speed of the particles.Besides such improvements in controlling particle flow and in flowcytometry instrumentation generally, significant progress has also beenmade with respect to other aspects of flow cytometry, for example, withrespect to the methods of cell preparation, new fluorescent dyes and newmarkers of cell properties.

These technological advances in flow cytometry have made it possible touse flow cytometers in a variety of areas. For example, flow cytometersare now used for analyzing white blood cells in AIDS patients. Also forexample, flow cytometers are now employed in performing cancer diagnosisand stem cell sorting. Indeed, flow cytometry is now widely recognizedas an important clinical and research tool. However, even though thesize of a flow cytometer has been reduced from a piece of equipmentoccupying an entire room to a table top system with ever increasingfunctionality and performance, flow cytometers continue to cost between$150K and $1M, and consequently remain a tool affordable only by majormedical centers and laboratories. Size and price reduction by orders ofmagnitude (e.g. 1000 times) are necessary to make flow cytometers aprevailing diagnosis tool that can be afforded by more hospitals andmedical practitioners around the world.

One technique that holds promise for miniaturizing flow cytometers isthe use of microfabricated flow cells, enabled by advances inmicrofluidics. Integrated microfluidic chips that perform a variety offunctions for chemical analysis and biological screening have found wideapplications in the pharmaceutical industry and have accelerated theprogress of research in biotechnology. Several research groups havedemonstrated the ability to manipulate cells and micro-particles inmicrofluidic devices using the effects of fluidic pressure,dielectrophoresis, optical trapping, and electro-osmosis. Moreparticularly, the introduction of microfabricated electrodes in thefluidic channels of microfluidic devices can facilitate the opticaldetection of particles by controlling and manipulating the positions,angles, and populations of the particles in microfluidic channels viathe dielectrophoretic effect.

There are several reasons that make these results particularly relevantto the development of compact flow cytometers. First, biological cellsizes fit well with the dimensions of the microfluidic devices that canbe easily and precisely fabricated using microfabrication techniquessuch as lithography and molding. Second, microfluidic devices tend tosupport laminar flow, making the flow control simpler and fluidtransport highly efficient. Third, micro-scale integration allows morefunctionality (e.g. pumps, valves and switches) to be incorporated intothe device. Finally, two-dimensional or even three-dimensional arraystructures can be fabricated to enhance the performance of the systemand alleviate the limit of device throughput.

Although rapid progress has been made in microfluidics that isapplicable to flow cytometry, the scheme of optical detection employedin flow cytometry has not experienced similarly important advances orchanges. In particular, while the hardware utilized in performingoptical detection has continued to evolve, resulting in more advancedlasers, more sensitive detectors, and superior optical mechanicalcomponents, there nevertheless has not been any paradigm shift in termsof the manner in which optical detection is performed in flow cytometry.As a result, the expensive and bulky optical setup currently necessaryfor fluorescence detection threatens to become a bottleneck restrictingthe realization of compact, low-cost flow cytometers. Additionally, therelatively high cost of lasers and light detectors for use in flowcytometry is further exacerbated when one reduces the size of theoverall system.

For at least these reasons, it would be advantageous if an improvedoptical detector, optical detection scheme or optical detection methodcould be developed for use in detecting small objects or particles suchas cells or DNA, as could be used for, among other things, performingflow cytometry and related techniques. More particularly, it would beadvantageous if, in at least some embodiments, such an improved opticaldetector/detection scheme/method could be designed in which smaller (interms of size and/or weight) optical components could be employed.Additionally, it would be advantageous if, in at least some embodiments,simpler, less expensive components could be employed for the purposes ofgenerating and/or sensing light.

BRIEF SUMMARY OF THE INVENTION

The present inventors have recognized the desirability of achievingimproved photonic designs and technologies for detecting small objectsor particles, for use in various applications such as flow cytometryinvolving the use of microfluidic components. For example, the presentinventors have recognized the desirability of providing a cost effectivesolution to the problem of fluorescence (and side scattering) detectionin flow cytometry and, more particularly, have recognized the importanceof at least one of (a) integrating optical components with fluidiccircuits to reduce the size and weight of the overall system, and (b)developing innovative architectures of photonic circuits to achievedesired levels of sensitivity without the need for expensive componentssuch as lasers (e.g., main frame lasers) and/or ultra sensitivedetectors (e.g., photomultiplier tubes (PMTs)).

In accordance with at least some embodiments of the present invention,to achieve such goals the present inventors propose amicrofluidic-photonic integrated circuit optical interrogation devicethat can be utilized as a microfabricated flow cytometer. The deviceincludes a photonic circuit integrated monolithically with themicrofluidic channels such that the optical interrogation zones are inthe proximity of and well aligned to the optical waveguides that collectthe fluorescence and/or scattering light signals. The use of such awaveguide approach to replace free-space optics eliminates the needs forlenses and precision mechanics for optical alignment, making significantsize and weight reduction possible. The device can be fabricated using afluidic-photonic integrated circuit (FPIC) process.

In at least some such embodiments, multiple waveguides are employed toform an array waveguide structure so that, along the direction of flow,a particle (e.g., an analyte, cell or segment of DNA) will pass a seriesof waveguide-defined optical interrogation zones, each producing asignal that is correlated in time and space to the others. In oneexample of such an embodiment, an array of eight parallel waveguides isemployed so that the signal produced by a particle will be detectedeight times. At the detection end, an array of eight detectors can beemployed or, alternatively, it is possible to combine the eightwaveguides into a single output waveguide and use only a single detector(or, also alternatively, more than one but less than eight detectors canbe employed). For a single detector approach, the signals from the eightwaveguides can be multiplexed in the time domain, with a time delay ofthe demultiplexed operation being set equal to the transit time of theparticle as it passes between adjacent waveguides.

In at least some embodiments, the present invention relates to a devicethat includes a fluidic channel capable of conducting a fluid containingat least one particle, a source of electromagnetic radiation arranged toprovide the electromagnetic radiation into the fluidic channel tointeract with the at least one particle contained within the fluid asthe fluid is conducted by the fluidic channel, and a first plurality ofoptical waveguides having respectively a plurality of ends positionedalong the fluidic channel. The optical waveguides receive at least someof the electromagnetic radiation after the electromagnetic radiation hasinteracted with the at least one particle.

Additionally, in at least some embodiments, the present inventionrelates to a fluidic-photonic integrated circuit (FPIC) device thatincludes a microfluidic channel, means for exciting a material withinthe microfluidic channel, and a first optical waveguide for receivingelectromagnetic radiation as a result of the exciting of the material.Information regarding the material is detected based upon the receivedelectromagnetic radiation.

Further, in at least some embodiments, the present invention relates toa method of manufacturing a fluidic-photonic integrated circuit (FPIC)device. The method includes casting pre-polymer onto aphoto-lithographically patterned mold, thermally-curing the pre-polymer,and demolding a first piece of thermally-cured polymer from the mold.The method also includes bonding the first piece to a second piece ofpolymer material to form a fluidic channel, and implementing the fluidicchannel in relation to a further structure capable of receiving andguiding electromagnetic radiation away from the fluidic channel.

Additionally, in at least some embodiments, the present inventionrelates to a method of obtaining information regarding at least oneparticle suspended within a flowing fluid. The method includes applyingincident light to the fluid and to the at least one particle suspendedwithin the fluid as the fluid flows through a fluidic channel, andguiding scattered or fluorescent light resulting from an interactionbetween the incident light and the at least one particle by way of aplurality of optical waveguides extending away from the fluidic channelto at least one detection device. The method further includes derivingat least one signal at the at least one detection device based upon theguided, scattered or fluorescent light, and performing a calculationbased upon the at least one signal resulting in the information, theinformation being indicative of at least one characteristic of the atleast one particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a) and 1(b) respectively provide a schematic, side perspectiveview and a top plan view of an exemplary integrated fluidic-photonicdevice having a fluidic channel, optical waveguide, and adielectrophoretic (DEP) cage, in accordance with at least someembodiments of the present invention;

FIGS. 2( a)-(c) illustrate steps of an exemplary process ofmanufacturing the device of FIGS. 1( a)-(b) having DEP electrodesintegrated with a microfluidic channel;

FIGS. 3( a) and (b) respectively show a schematic view and a sidecross-sectional view of an exemplary fluidic-photonic integrated circuitdevice that can be employed in an improved optical detector inaccordance with at least some embodiments of the present invention;

FIG. 4 shows a side cross-sectional view of an alternate embodiment ofthe fluidic-photonic integrated circuit device of FIGS. 3( a) and (b) inaccordance with some other embodiments of the present invention;

FIGS. 5( a)-(b) show steps of an exemplary process that can be employedto fabricate one or more fluidic channels employed in the devices ofFIGS. 3( a)-(b) and 4;

FIG. 6 shows in schematic form an improved optical detector inaccordance with at least some embodiments of the present invention,where the detector employs a fluidic-photonic integrated circuit similarto that of FIGS. 3( a)-(b);

FIGS. 7( a) and (b) are graphs showing exemplary output signals from asingle output of the waveguide demultiplexer of the fluidic-photonicintegrated circuit of FIGS. 4( a)-(b);

FIGS. 8( a)-(c) are graphs showing exemplary time variation of photoncounts intensity generated from eight waveguide outputs of an alternateembodiment of the fluidic-photonic integrated circuit of FIG. 4 wherethe data in (a-c) were obtained from fluorescent beads of decreasingsize and fluorescence intensity;

FIG. 9 is a schematic illustration of time domain cross-correlation;

FIG. 10 is a flow chart illustrating steps of an exemplary iterativecross-correlation process;

FIGS. 11( a) and (b), respectively, are graphs showing exemplarycross-correlated signals obtained using the raw data of FIGS. 8( b) and(c), respectively; and

FIGS. 12( a) and (b), respectively, are graphs showing exemplary signalchains obtained using the raw data of FIGS. 7( a) and (b), respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As described in detail below, the present invention is intended toencompass a variety of different embodiments of microfluidic-photonicintegrated circuits and similar devices, as well as systems thatimplement those integrated circuits and devices. Such devices can beemployed for a variety of purposes including, for example, to detect thepresence of biological particles such as cells and DNA or otherparticles, and/or in various applications such as flow cytometry andother techniques. Additionally, the present invention is intended toencompass various methods of operating and manufacturing such integratedcircuits and other devices (and/or systems that implement thoseintegrated circuits and devices). In at least some embodiments, forexample, microfluidic-photonic-dielectrophoretic integrated circuits canbe fabricated by way of a process involving micro-molding, polymerbonding, and channel waveguides with capillary filling. At least some ofthe circuits described herein can be considered to represent a new classof circuits particularly attractive to lab-on-a-chip and biomedicalapplications.

Referring to FIGS. 1( a) and 1(b), a schematic, side perspective viewand a top plan view are provided of a first exemplarymicrofluidic-photonic integrated circuit device 10 that is capable ofallowing fluorescent excitation and detection to be performed. Althoughcapable of being employed in various environments, the device 10 in atleast some cases is intended to perform on-chip optical detection ofbiological particulate material or analytes such as single cell(s)and/or small aggregations of DNA. As shown particularly in FIG. 1( a),the device 10 includes first and second waveguides 12 and 14,respectively, that are aligned with one another and extend away fromeach other. Further as shown, the first and second waveguides 12, 14respectively extend up to opposite sides of a fluidic channel 16 thatextends perpendicularly between the waveguides. Light passing throughthe first waveguide 12 along a direction generally indicated by an arrow13 passes through a target region 17 of the fluidic channel 16, where itcan interact with particulate material 19 flowing through the targetregion. Some or all (or possibly none) of the light, depending upon thelight's interaction with the particulate material, then passes throughthe second waveguide 14 along a direction generally indicated by anarrow 15.

Additionally, the device 10 also includes a dielectrophoretic electriccage 18 formed by four pairs of diagonally-arranged electrodes 20, 22,24 and 26, respectively, where each pair of electrodes includes an upperand a lower electrode as illustrated in FIG. 1( a). Thedielectrophoretic electric cage 18 is designed to trap and rotate thetarget particular material electrically. The trapped object tends toreside at the position in the fluid where the potential energy islowest. If an ac voltage is applied to the four pairs of electrodes 20,22, 24 and 26 with a phase difference (e.g., with approximately90-degree phase differences between each adjacent pair of electrodes),then the trapped object can obtain angular momentum and spin while it istrapped.

Referring additionally to FIG. 1( b), the top view of the device 10further illustrates the relative arrangement of the integrated pairs ofelectrodes 20, 22, 24 and 26, the waveguides 12, 14 and the fluidicchannel 16. As shown, each of the electrodes 20, 22, 24 and 26 extendsat 45 degree angles relative to its respective neighboring one of thewaveguides 12, 14 and relative to the fluidic channel 16, so as to forman “X” arrangement overlapping a “+” arrangement formed by thewaveguides and fluidic channel. Also as shown, the electrodes 20, 22,24, and 26 protrude slightly into the target region 17 of the fluidicchannel 16. It should be noted that the fluidic channel 16, waveguides12, 14 and electrodes 20, 22, 24 and 26 in the present embodiment aresmall or microscopic in size, typically having a smallest feature sizebetween 5 and 50 micrometers.

A variety of processes can be implemented in order to manufacture orotherwise form the device 10 of FIGS. 1( a)-(b). Referring to FIGS. 2(a)-(c), steps of one exemplary process for fabricating the device 10 areshown. In particular, a self-aligned process is employed to form thefluidic channel 16 and waveguides 12, 14 between the four pairs ofelectrodes 20-26 in a manner such that the electrodes of each pair areparallel to one another. As shown in FIG. 2( a), in a first step thelower electrode of each pair of the electrodes 20-26 are all formed atopa polydimethylsiloxane (PDMS) substrate 29 (more particularly, twoelectrodes from the first and second pairs of electrodes 20 and 22 areshown). Atop the four lower electrodes are then positioned thewaveguides 12, 14, and atop the waveguides is positioned a handle wafer31. The target region 17 of the fluidic channel 16 is within the spaceformed by the adjacent electrodes and waveguides. In the presentembodiment, the electrodes are made from gold (Au), albeit othermaterials can be used in alternate embodiments.

Once the structures have been assembled as shown in FIG. 2( a), and inparticular once the lower electrodes have been formed by way of thatstep, the handle wafer 31 is delaminated from atop the waveguides 12,14, at a step shown in FIG. 2( b). The removal of the handle waferallows for the upper electrodes of each of the pairs of electrodes to beformed above the waveguides 12, 14. It is desirable that the upperelectrodes and lower electrodes of each pair of electrodes 20-26 bealigned (e.g., parallel) with each other, and that the set of four upperelectrodes and the set of four lower electrodes be respectively formedon two different planes on both (opposite) sides of the fluidic channel.To achieve such alignment, the addition of the upper electrodes of eachpair, which can be formed within an electrode-patterned substrate caninvolve the use of an optical mechanical alignment tool such as acontact mask aligner or a wafer bonder. FIG. 2( c) shows the upperelectrodes to be assembled atop the waveguides 12, 14, and additionallyshows another PDMS substrate 29 to be positioned atop the upperelectrodes. Thus, the target region 17 is entirely enclosed within thewaveguides 12, 14, the pairs of electrodes 20-26 (only two pairs ofwhich are shown in FIG. 2( c)), and the PDMS substrates 29, and thetarget region in particular has four pairs of electrodes around itforming the dielectrophoretic electric cage 18.

Turning to FIGS. 3( a)-(b) and 4, additional embodiments of exemplaryfluidic-photonic integrated circuit (FPIC) devices differing from thatof FIGS. 1( a)-(b) are shown. The devices shown in FIGS. 3( a)-(b) and 4in particular are guided wave photonic circuit devices that are moresophisticated in their operation than the device of FIGS. 1( a)-(b).Further, as with the device 10 of FIGS. 1( a)-(b), the devices shown inFIGS. 3( a)-(b) and 4 are fabricated to include microfluidic channels.Through the use of such microfluidic channels, the devices of FIGS. 3(a)-(b) and 4 can take a miniaturized form and are capable of deliveringnew functions including, for example, functions relating to theperformance of flow cytometry, which is the workhorse for manybiomedical applications. In particular, the devices allow for flowcytometry with relatively high signal reliability and sensitivity to beachieved, notwithstanding possible non-uniformities in the biologicalsamples (or other sensed material) and/or complex flow patterns. Inalternate embodiments, microfluidic channels need not be employed.

FIG. 3( a) in particular shows a schematic diagram of a first FPICdevice 40 that can be employed in an improved optical detector. FIG. 3(b) additionally shows a side cross-sectional view of a portion of thedevice 40 (e.g., in cut-away), while FIG. 4 shows a side cross-sectionalview of a portion (e.g., in cut-away) of an alternate embodiment of thedevice 40, namely, a device 60. As shown, each of the devices 40, 60 ofFIGS. 3( a)-(b) and 4 includes a respective fluidic channel 50, 70including a respective vertical section 48, 68. As shown particularly inFIG. 3( a), the fluidic channel 50 of the device 40 extends between afirst fluidic inlet/outlet 33 and a second fluidic inlet/outlet 34(although not shown, the fluidic channel 70 of FIG. 4 also extendsbetween two such inlets/outlets). Although the sections 48, 68 are shownto be vertically-oriented, the sections need not be oriented in thismanner and instead could take on other orientations, such as horizontalorientations.

In addition to the fluidic channels 50, 70 and associated verticalsections 48, 68, each of the devices 40, 60 includes a respective firstexcitation waveguide 42, 62 and a respective second excitation waveguide44, 64. The respective excitation waveguides of each respective device40, 60 are aligned with, and extend in opposite directions from oppositeends of, the respective vertical section 48, 68 of the respectivedevice. Thus, the waveguides 42, 44 are each aligned with the verticalsection 48, with the first waveguide 42 extending upward away from thetop of the vertical section and the second waveguide 44 extendingdownward from the bottom of the vertical section. Likewise, thewaveguides 62, 64 are each aligned with the vertical section 68, withthe first waveguide 62 extending upward away from the top of thevertical section and the second waveguide 64 extending downward from thebottom of the vertical section.

The waveguides 42, 44 of the device 40 and the waveguides 62, 64 of thedevice 60 each perform a similar function to that performed by thewaveguides 12, 14 of the device 10 of FIGS. 1( a)-(b), namely, to causeexcitation light to be directed toward (and possibly away from) a targetregion, where the target regions in these embodiments are the verticalsection 48 and the vertical section 68, respectively. More particularly,the two waveguides 42, 44 and 62, 64 near the ends of the respectivevertical sections 48, 68 of the respective fluidic channels 50, 70deliver optical power for fluorescent excitation. The excitation lightcan come from various directions including from the top or the bottom ofthe device (e.g., as shown in FIGS. 3( a)-(b) and 4, similar to amicroscope setup in terms of the direction of light passage through themicroscope lens) as well as, in alternate embodiments, from otherdirections. The waveguides 42, 44, 62 and 64 as integrated on thedevices 40, 60 in particular are able to provide convenient access ofthe excitation light at chosen wavelengths and in well-definedexcitation directions. The well-defined excitation directions achievedthrough use of the waveguides 42, 44, 62 and 64 in particular facilitatethe measurements of forward, side, and back scatterings of light as itencounters the sample particles or analytes flowing through the verticalsections 48, 68 of the fluidic channels 50, 70.

In addition to the respective pairs of excitation waveguides 42, 44 and62, 64, each of the respective devices 40, 60 also includes a respectivefirst array of horizontal waveguides 52, 72 and a respective secondarray of horizontal waveguides 54 and 74, respectively. The waveguidesof the respective first and second arrays 52 and 54 respectively arearranged oppositely one another on left and right sides of the verticalsection 48, and extend horizontally in opposite directions away fromthat vertical section, while the waveguides of the respective first andsecond arrays 62 and 64 respectively are arranged oppositely one anotheron left and right sides of the vertical section 68, and extendhorizontally in opposite directions away from that vertical section.More particularly as shown, in the present embodiments, each of thearrays 52, 54, 72 and 74 has eight waveguides that extend parallel toone another horizontally away from the respective vertical section 48,68, with each waveguide of each array being spaced apart from theneighboring waveguide(s) of the respective array by a predeterminedamount of distance (e.g., 100 micrometers between the centers ofneighboring waveguides). For each of the waveguides of the left-sidearrays 52 and 72, there is a corresponding waveguide in the respectiveright-side array 54 and 74 that is aligned with that left-side arraywaveguide.

Each of the waveguides of the arrays 52, 54, 72 and 74 is capable offunctioning as a detection waveguide capable of conducting/guiding lightemanating from a respective one of the vertical sections 48, 68, andthus serves to allow for optical interrogation. In the presentembodiments, optical detection occurs by sending excitation light intothe vertical sections 48, 68 by way of one or both of the excitationwaveguides 42, 44, 62, 64 associated with the respective verticalsection, allowing that light to interact with and be scattered by theliquid-suspended sample particle(s) (e.g., cells, DNA or microparticles)flowing through the respective vertical section, and then sensing theamounts of scattered light that are received within and transmitted bythe waveguides of the arrays 52, 54, 72 and 74. The light detected fromthe waveguides of the arrays 52, 54, 72 and 74 thus is indicative of theliquid-suspended sample particles (e.g., cells, DNA or microparticles)flowing through the respective fluidic channels 50, 70. As will bedescribed in further detail below, the detection of light by way ofmultiple detection waveguides rather than merely one detection waveguideis particularly advantageous.

The dimensions of the fluidic channels 50, 70, the excitation waveguides42, 44, 62 and 64 and the detection waveguides of the arrays 52, 54, 72,and 74 can vary depending upon the embodiment. In the presentembodiments, the cross-sectional dimensions of the fluidic channels 50,70 and particularly the vertical sections 48, 68 of those channels is50×50 μm², although other cross-sectional dimensions are also possible.Additionally, the cross-sectional dimensions of each of the excitationwaveguides 42, 44, 62 and 64 as well as each of the waveguides of thearrays 52, 54, 72 and 74 in the present embodiments further are 50×50μm², although other dimensions are also possible. While in at least someembodiments, including the present embodiments, it is desirable that thewaveguides have substantially the same cross-sectional dimensions (andshapes) as the corresponding fluidic channels, this need not be thecase. Further, in the present embodiments of FIGS. 3( a)-(b) and 4, boththe excitation waveguides 42, 44, 62, 64 and the detection waveguides ofthe arrays 52, 54, 72, 74 are multi-mode devices with a numericalaperture of 0.3.

The use of the arrays 52, 54, 72 and 74 of waveguides results isadvantageous in several regards. Because each of the arrays 52, 54, 72and 74 has eight parallel spaced-apart waveguides, each device 40, 60has a capability of detecting a particle eight times as it passesthrough the respective vertical section 48, 68. More particularly, theuse of the eight waveguides of each of the arrays 52, 54, 72 and 74, byproviding eight detection points for the same target flying by, resultsin detected signals that have improved signal-to-noise ratios comparedwith conventional optical detection systems and in which randomnesscaused by Brownian motions is suppressed. In addition, the use of thesearrays of waveguides allows for the performing of time-of-flightmeasurements, to determine the velocity of particles flying by, as wellas allows for multi-label fluorescent detection with wavelength filters.Compared with single-point detection, the data that can be obtainedusing such multiple sampling points can provide rich information aboutthe properties of a particle (or particles) under scrutiny, as well asthe particle's interplay with the fluid, and the statistic behaviorsunder Brownian forces.

As discussed above, on the left sides of the respective verticalsections 48, 68 of the respective fluidic channels 50, 70 are positionedrespective left-side arrays 52, 72, each of which has eight detectionwaveguides with separated outputs. Likewise, on the right sides of thosevertical sections 48, 68 are positioned respective right-side arrays 54,74, each of which also has eight detection waveguides. However, whilethe right-side array 54 of FIG. 3( b) has eight separated outputsassociated with its respective eight detection waveguides, theright-side array 74 of FIG. 4 rather includes an 8×1 waveguide combineror demultiplexer 76 such that all of the eight waveguides of that arrayeventually are merged to form a single output waveguide. That is, whilethe array 74 of the device 60 includes eight horizontal detectionwaveguides extending away from the vertical section 68, these waveguideseventually bend toward one another and are merged/joined with oneanother as they proceed farther away from the vertical section.

FIG. 4 in particular shows how four adjacent pairs of the eightwaveguides of the array 74 bend and merge with one another to form anarray of four waveguides 77. However, it should further be understoodthat FIG. 4 only shows the portion of the demultiplexer 76 in whicheight waveguides are merged into four waveguides, and that thedemultiplexer additionally involves the merging of those four waveguidesinto two waveguides and then subsequently into a single outputwaveguide. During operation of the device 60, the demultiplexer 76receives time-multiplexed signals from eight detection zonescorresponding to the eight waveguides of the array 74. As described infurther detail below in relation to FIG. 7( a) et seq., through the useof the information obtained from the array 74, the 8×1 demultiplexer 76is able to generate an overall signal that includes all of theinformation obtained from the eight detection waveguides, and to restorethe time domain signal chain.

Although both the devices 40 and 60 of FIGS. 3( a)-(b) and 4 are capableof achieving enhanced detection sensitivity through the use of themultiple detection waveguides in their respective waveguide arrays 52,54, 72 and 74, the use of the demultiplexer 76 in the device 60 makes itpossible to reduce the number of detectors receiving the lightcommunicated by way of the waveguides to only one detector (or at leastto a number of detectors less than the number of waveguides that areinterfacing the fluidic channel). Thus, use of the demultiplexer 76allows for a savings of the hardware costs associated with havingmultiple detectors (albeit at an expense of device throughput).

The detection sensitivity achieved by the devices 40, 60 is dependentupon the number of detection waveguides of the arrays 52, 54, 72 and 74.This is true both whether the demultiplexer 76 is employed or notemployed (where the demultiplexer is not employed, as discussed infurther detail below, the multiple signals provided by the multiplewaveguides of each array can be used to perform cross-correlationoperations so as to achieve enhanced signal-to-noise ratios). Indeed,the detection sensitivity can be enhanced by increasing the number ofwaveguides of the array 74 and/or the space-demultiplexed waveguidestructure. Nevertheless, in the present embodiments of FIGS. 3( a)-(b)and 4, with a total of eight waveguide detection channels/zones, thedetection sensitivity can be enhanced by nearly 1,000 times incomparison with that afforded by a single channel device. This manifestsone advantage of the FPIC approach over free-space optical set-ups usedin performing conventional flow cytometry because for the latter, thenumber of interrogation zones is significantly limited by space andcost.

The fluidic-photonic integrated circuit (FPIC) devices 40, 60 in theembodiments of FIGS. 3( a)-(b) and 4 are made entirely (orsubstantially) of polymer such as PDMS, and are fabricated by way ofmicro-molding and waveguide capillary filling. Details of themicro-molding process that can be utilized to create the devices 40, 60of FIGS. 3( a)-(b) and 4 are illustrated in FIG. 5( a)-(b). As shown inFIG. 5( a), a soft lithography process can be employed to fabricatemicrofluidic channels such as the channels 50, 70 discussed above. Asshown, a semiconductor or glass wafer 150 is provided at a step 152, andthen at a step 154 a spin-on thick resist 156 is added to the wafer toform a combined structure 158. Further, at a step 160, the combinedstructure is exposed to ultraviolet light 162 by way of a mask (notshown). Subsequent to the photo-lithographic patterning step 160, thecombined structure 158 is now a modified combined structure 165 having amodified (patterned) thick resist layer 167. The modified combinedstructure 165 can also be referred to as a “mold master”.

Subsequent to the step 160, the modified combined structure 165 is thenbaked by way of a thermo-curing process, for example, thermo-curing at65° C. for 4 hours. Then, at a step 166, a pre-polymer layer 168 is castand cured upon the modified combined structure 165. Then, at a step 170,the pre-polymer layer 168 is peeled from the mold master, thustransferring the pattern to the pre-polymer layer. In at least someembodiments, the mold master is made from photo-lithographicallypatterned SU-8-50 photoresist (such as that available from MicroChem,Inc. of Newton, Mass.) on a 4″ silicon wafer (such as that availablefrom Silicon Quest International, Inc. of Santa Clara, Calif.). Also,the pre-polymer layer 168 cast onto the mold master can be a PDMS layersuch as Gelest OE 41 available from Gelest, Inc. of Morrisville, Pa.

The steps shown in FIG. 5( a) result in the creation of the pre-polymer(PDMS) layer 168 that has channel patterns 172 complementary to thepatterns of the modified thick resist layer 167. Referring additionallyto FIG. 5( b), in order to make an enclosed microfluidic channel, thepre-polymer layer 168 is in turn bonded to another pre-polymer (e.g.,PDMS) layer 174. Typically, both of the pre-polymer layer 168 and 174will have the same refractive index (e.g., 1.407). A short treatment(e.g., 10 seconds) of high power (e.g., 100 Watts) oxygen plasma (e.g.,using the Technics 500-II Plasma Etcher and Asher System) can be used toactivate the surfaces of the pre-polymer layers 168, 174 to facilitatepermanent bonding of those layers, thus completing the fabrication ofone or more microfluidic channels corresponding to the patterns 172. Inalternate embodiments an ultraviolet/ozone treatment can be employedinstead of the oxygen plasma treatment to achieve bonding.

Various similar molding techniques can also be used to fabricate ridgewaveguides with a chosen polymer of an appropriate refractive index,which can be employed as the waveguides 42, 44, 62, 64 or the waveguidesof the waveguide arrays 52, 54, 72 and 74 described above. One suchtechnique that can be employed to make the waveguides is achannel-waveguide filling process. To make channel waveguides by way ofthis technique, some chosen channels are filled with a polymer of higherrefractive index, for example, Gelest OE 42 PDMS (also available fromGelest Inc.) can be chosen as the core material. Pre-polymer isintroduced into the channels through the inlets. Pre-polymer cancompletely fill the channels in a short period of time, e.g., 20minutes. Then, upon performing the same thermo-curing procedure asmentioned before, the core material is solidified and takes on a desiredrefractive index (e.g., 1.42). It should be further noted that, ifstriations at the end of a waveguide are caused due to waveguidecutting, these can be removed by polishing; at the same time, facetpolishing does not appear to be usually necessary since the striationshave not appeared to disturb experimental signal measurements.

For the purpose of realizing monolithic fluidic-photonic integratedcircuits (FPICs), several integration schemes can be utilized. Forexample, for high sensitivity chemical sensors that require longinteraction length, one can employ an integrated structure having asingle-mode ridge waveguide inside or aligned with the microfluidicchannel so that the light wave propagates in the same direction as theflow. More particularly, to achieve such an integrated structure, boththe waveguide and the microfluidic channel are formed in the samefabrication process, with the waveguide being formed by creatingpassage(s) with walls of a first, lower index of refraction that aresubsequently filled with a liquid that, upon being solidified by heat orUV treatment, takes on a second, higher index of refraction. Themicrofluidic channel can have the same cross-sectional dimensions as thewaveguide, or the two structures can have different cross-sectionaldimensions, as desired. Also, for applications where on-chip opticalprocessing or contact-free detection is needed, one can also employ astacking structure in which waveguides and fluidic channels are locatedat different planes so that the waveguides and fluidic channels can berouted without crossing one another.

Still another manner of integrating waveguides and fluidic channels is aself-forming technique that utilizes a capillary effect andimmiscibility between liquids inside microfluidic channels to createwaveguides that intersect the microfluidic channels. Such an integrationscheme is particularly suitable for highly-localized fluorescentexcitation and detection. For the self-forming process, a fluidicchannel and a waveguide channel (or multiple waveguide channels) areformed that intersect with one another. The fluidic channel is thenfilled up with BSA (protein) aqueous solution before liquid(pre-polymerized) PDMS is provided to fill the waveguide channel(s) thatintersect the fluidic channel. Since the liquid PDMS and the BSAsolution are immiscible, the liquid PDMS will not enter the fluidicchannels even though they intersect. After the liquid PDMS is thermally(e.g., at 60 degrees C.) or UV cured to become solid PDMS, the BSAsolution is removed from the fluidic channel, yielding an array ofwaveguides in very close proximity to the fluidic channels for efficientlight coupling. In at least some embodiments of FPICs to be used forflow cytometry, a waveguide/channel integration structure similar to butsimpler than what is used in the self-forming technique can also beemployed.

Notwithstanding the above discussion, the present invention is intendedto encompass a variety of FPIC devices having features, or beingfabricated by way of techniques, other than those mentioned above. Forexample, in alternate embodiments, FPIC devices can have any number ofdetection waveguides corresponding to the arrays of waveguides 52, 54,72 and 74, including more than eight or less than eight waveguides ineach array (or even only one waveguide on each side of the fluidicchannel). Likewise, although the waveguides of the arrays 52, 54, 72 and74 are perpendicular to the vertical sections 48, 68 of the fluidicchannels, in alternate embodiments, the waveguide(s) can approach thefluidic channels at oblique or other angles. Curved waveguide surfacescan also be formed to create light focusing effects to either increasethe numerical aperture of the waveguides or to move the waveguidesfurther away from the fluidic channels. It should further be noted that,while in at least some embodiments such as those discussed above, theFPIC devices are PDMS-based microchips, in alternate embodiments theFPIC devices can be made from other materials and via other processesthan are used to develop PDMS-based microchips.

Referring to FIG. 6, an exemplary flow cytometry system 80 employing aFPIC device 100 similar to the device 40 of FIGS. 3( a)-(b) (e.g.,without any demultiplexer) is shown in schematic form. As shown, theFPIC device 100 includes the microfluidic channel 50 having the verticalsection 48 and the first and second inlet/outlets 33, 34, as well as theexcitation waveguides 42 and 44. However, in this embodiment (contraryto that of FIGS. 3( a)-(b)), only one of the waveguide arrays 52, 54(namely, the right-side array 54) is employed and, as a result, signalsare only detected along one side of the microfluidic channel 50. Furtheras shown, the first inlet/outlet 33 functions as an inlet and receivespumped fluid from plastic tubing 81, by which the inlet is coupled to apumping unit 82. The second inlet/outlet 34 in turn functions as anoutlet that in the present schematic diagram is shown to be left openbut which typically is coupled to a receptical such as a waste beaker(e.g., in an experimental set-up) or can be coupled back to the pumpingunit 82. As part of the standard microfluidic device fabrication andpackaging process, appropriate connectors can be fabricated to connectthe plastic tubing 81 to the inlet 33 on the chip without leakage. In atleast some embodiments, a syringe pump can be used as the pumping unit82 to deliver liquid samples.

Also, in the present embodiment, laser excitation light from a source 84is coupled by way of multi-mode optical fiber 86 to one of the twowaveguides 42, 44 (in this case the waveguide 44) that are aligned withand face the respective ends of the vertical section 48 of the fluidicchannel 50. In the present embodiment, to secure the connection betweenthe optical fiber 86 and the waveguide 44, a multi-mode fiber isinserted into the waveguide channel prior to filling of that channelwith pre-polymerized PDMS and solidification of that PDMS (before thecore material of the waveguide is solidified) such that, after PDMScuring and solidification, an encapsulated fiber-waveguide structure iscreated. Such an encapsulated fiber-waveguide structure is capable ofshowing low insertion loss (<0.3 dB) and negligible interfacereflection, as well as mechanical robustness. Further as shown in FIG.6, the light transmitted by the detection waveguides of the array 54away from the fluidic channel 50 is directed toward an objective lens88, which in turn provides that light to a CCD camera 90. A near-fieldimage thus is formed on a CCD camera screen of the camera 90.

Depending the upon the embodiment, the camera 90 or another device incommunication with the camera (not shown) can include a processingdevices that receive signals from the camera, allowing for furtherprocessing operations to be performed, some of which are described infurther detail below. The processing can be, for example, amicroprocessor, programmable logic device or integrated circuit devicesuch as a digital signal processing (DSP) chip or other processingdevice. In at least some embodiments, the processing device can be partof, or assume the role of, a control device or controller capable notonly of processing information but also capable of generating andcontrolling the output of (e.g., on a display or onto a network, such asthe internet) signals, information, or data. In some such embodiments,the controller also is capable of monitoring and/or controlling otherdevices/components of the flow cytometry system 80 such as the pumpingunit, the excitation laser, and/or other devices/components. Further, instill other embodiments, a processing device and/or controller can becoupled to receive information from the detection waveguides via adevice other than the camera 90. It should further be understood thatembodiments of the invention not being employed for the purpose of flowcytometry also can employ a processing device or controller similar tothat described above. Additionally, it should be understood that theprocessing device/controller should be generally understood to encompassone or more memory devices or computer-readable storage media capable ofgoverning operation of the processing device/controller.

Although not shown in FIG. 6, a demultiplexer such as the demultiplexer76 can also be employed as part of (or in conjunction with) the array ofwaveguides 54. As mentioned above, the 8×1 demultiplexer 76 of FIG. 4serves to reduce the number of optical detectors needed to receive thelight from the array of waveguides 74. Also, the demultiplexer 76 allowsfor the restoring of a time-domain signal chain from noise-masked data.Referring to FIGS. 7( a)-(b), exemplary output of a 8×1 demultiplexersuch as the demultiplexer 76 of FIG. 4 in response to detected signalsis provided to illustrate such operation of the demultiplexer. FIG. 7(a) in particular shows data that was obtained using a sample containing5 μm fluorescent microbeads, while FIG. 7( b) in particular shows datathat was obtained using a sample containing 1 μm fluorescent microbeads.The data represents the combination of the signals from 8 separatechannels (e.g., the combination of all of the eight waveguides of thearray 74).

When the detected output signal is weak, the raw data shown in FIGS. 7(a) and (b) can be largely corrupted by noise due to stray light and theelectronic noise of the CCD camera. Nevertheless, in spite of thepotentially poor signal quality, it is possible to perform the followingalgorithm to restore the signal chain in the time domain, utilizing theproperty that the signals detected at the eight sequential detectionzones provided by the eight waveguides of the array 74 aretime-correlated. Specifically,

S(t)=f ₁(t)*f ₂(t−T)*f ₃(t−2T)*f ₄(t−3T)*f ₅(t−4T)*f ₆(t−5T)*f ₇(t−6T)*f₈(t−7T)  (1)

where S(t) is the time-dependent signal and T is the time interval for aparticle to pass through two adjacent waveguide channels (e.g., to passfrom one of the waveguides 74 to a neighboring one of the waveguides74). The value of T can be obtained from the time-of-flight measurement.

Thus, using Equation (1), the time dependent signal S(t) is determinedbased on a concept of time-correlation among the detection waveguides,as represented by multiple signals f_(i)(t). Additionally, it should benoted that, to remove the effects of high background and baseline drift,it is advisable to have each signal f_(i)(t) be passed through ahigh-pass filter before performing the operation according to Equation(1). Further, since all of the various f_(i)(t) signals represent signalintensity, negative values will be removed. For instance, should f₁happens to show a negative value at a certain time “t”, then the signalS(t) at the time t will become the product of the remaining 7 terms andits final value will be normalized by the power of 8/7. In other words,the normalized signal in the above case at the particular time becomesS^(8/7).

Turning to FIG. 8( a), as mentioned above, the arrays of eight parallelwaveguides such as the array 54 of FIGS. 3( a)-(b) also can be used toperform time-of-flight measurements. More particularly, when a beadtravels through the interrogation region (e.g., within the verticalsection 48 of FIG. 4), its fluorescence is detected by each of the eightwaveguides of the array 54 sequentially. The output intensity of each ofthe eight waveguides is recorded by the CCD camera 90. FIG. 8( a) showsexemplary intensities of the signals provided by the eight waveguidechannels as functions of time, which were obtained using an experimentalset-up employing 10 μm-diameter fluorescent beads. The center-to-centerdifference between intensity peaks (T) is the time period when a beadparticle travels across two adjacent waveguides. Knowing the distancebetween the centers of adjacent waveguides of the array 54, the velocityof the bead particle can be easily obtained. It should be further notedthat, in the case of 10 μm fluorescent beads, images of highsignal-to-noise ratio can be obtained directly from the output of anysingle waveguide.

Referring to FIGS. 8( b)-(c), to demonstrate the ability of sensitivityenhancement with the array waveguide structure, exemplary time-of-flightmeasurements were also performed using fluorescent beads of smallerdiameters than the 10 μm beads that were the basis of FIG. 8( a),namely, 5 μm (FIG. 8( b)) and 1 μm (FIG. 8( c)). Because thesefluorescent micro-beads have fluorescent dye doped over their entirevolumes, the fluorescent intensity of each respective bead isproportional its volume, making the fluorescence intensity eight timesand one-thousand times weaker than that of the 10 μm beads. FIGS. 8( b)and 8(c) respectively demonstrate that the directly-detected signals atthe output of each waveguide channel resulting from the 5 μm beads and 1μm beads, respectively, can be obscured and masked by the noise. Suchoutput from any single channel, analogous to the signal obtained from aconventional flow cytometer using a low power excitation source and alow sensitivity detector, cannot produce any meaningful signal (it isfor that reason that conventional flow cytometry systems require highpower lasers and photomultiplier tubes with photon counting sensitivity,which are expensive and non-scalable in size).

FIGS. 8( b)-(c) demonstrate that it can become more difficult to achievedesired signal-to-noise ratios as the particles to be sensed becomesmaller. In these circumstances, the methodology described above withrespect to Equation (1) for restoring a signal from noisy measurementsmay be inadequate for achieving output signals having desiredsignal-to-noise ratios. In particular, although Equation (1) provides amethod that is mathematically simple, a more robust method to restorethe real signal produced by each passing particle/analyte may be useful.In accordance with additional embodiments of the present invention, onesuch more robust method for providing output having improvedsignal-to-noise ratios is an additional multi-channel detectiontechnique that involves cross-correlation analysis. This calculationassumes that signals are correlated to beat between the differentwaveguides of a waveguide array such as the array 54, and takesadvantage of the knowledge that the true (light output) signals aretime-correlated while the noise is not.

The concept of cross-correlation is further illustrated graphically inFIG. 9. As illustrated, when a given particle passes through thevertical section 68 of the fluidic channel 50, first and second signals151 and 152 will be generated at two of the neighboring detectionwaveguides of the array 54. As long as the signals 151, 152 from the twochannels are time correlated, it is possible to obtain the time delay τbetween the two signals by calculating a cross-correlation function R(τ)defined in Equation (2):

R(τ)=ƒf1(t)*f2(t−τ)dt  (2)

where f1 and f2 are normalized intensity functions of two individualchannels and τ is a time domain variable. The cross-correlation functionR(τ) (also shown in FIG. 9) that maximizes τ becomes equal to thetime-delay between the two signals.

The above-described cross-correlation function can be extended forimplementation in relation to an array having an arbitrary number ofdetection waveguides. For example, with respect to the FPIC device 100in the above-described embodiment of FIG. 6 that has eight waveguidechannels, the above analysis can be extended to calculate aneight-channel cross-correlation, as follows:

R(τ)=f1(t)f2(t−τ)f3(t−2τ)f4(t−3τ)f5(t−4τ)f6(t−5τ)f7(t−6τ)f8(t−7τ)dt  (3)

In comparison with Equation (2) with its two terms, this eight-termmultiplication further serves to amplify the signal and suppress thenoise. It should further be noted that, with respect to Equations (2)and (3), it is not necessary to assume that every particle/analytetravel at exactly the same speed as is assumed in Equation (1), sincethe application of Equations (2) and (3) involves the calculating of thetravel velocity and time delay of each passing particle/analyte.

In view of the above considerations, use of an FPIC device/system (andespecially a microfluidic FPIC) such as the FPIC device 100 and system80 of FIG. 6 can be particularly advantageous when implemented inconjunction with cross-calculation techniques. Although thecross-correlation calculations according to Equations (2) and (3) aremore computation heavy than those according to Equation (1), thecalculations according to Equations (2) and (3) alleviate therequirement for keeping all of the particles/analytes in the streamlineof a constant velocity, thus greatly simplifying the design andprocessing complexities of fluidic channels. Further, because the FPICdevices can accommodate essentially any number of detection waveguidechannels without increasing the cost and complexity of the systemsubstantially, the advantages in signal quality achievable using thecross-calculation techniques are fully realizable.

Additionally, although the cross-correlation calculations according toEquations (2) and (3) when performed in the manner described above canbe relatively computation intensive, a further time domain spectroscopydetection process shown by a flow chart 200 in FIG. 10 can reduce thecomputational intensity of cross-correlation. In particular, as shown inthe flow chart 10, upon starting the flow chart at a step 202, theprocess begins at a step 204 by obtaining the signal information fromthe waveguides of the waveguide array (e.g., by way of a CCD camerareceiving signals from the waveguides of the array 54 of FIG. 6). Then,rather than calculating the time delay τ associated with the movement ofparticles between neighboring waveguides of the waveguide array, at astep 206 a value for the time delay is assumed. The time delay τ inparticular can be thought of as representing a range of times centeredabout a center time that is τ (e.g., τ=0.5 ms+/−0.5 ms). Subsequently,at a step 208, the cross-correlation algorithm (e.g., Equation (3) foran eight-waveguide array) is applied to the signal information and, at astep 210, it is determined whether the result from performing thisoperation is zero (or substantially zero, e.g., negligible).

If the result is not zero (or substantially below a set thresholdvalue), then at a step 212 a new value is assumed for the time delay,and step 208 is re-performed given that new value. The newly-assumedvalue of the time delay τ is typically an adjacent, incremental valuerelative to the previously-assumed value (e.g., given the first assumedvalue mentioned above, the next assumed value would be τ=1.5 ms+/−0.5ms). Given that the step 212 cycles back to the step 208, the steps 208,210 and 212 can be repeated iteratively as long as successivecross-correlation calculations produce non-zero results. However, oncethe result at step 210 is determined to be zero (or substantially zero),then it is further determined at a step 212 whether that has occurredalready for a number of (e.g., N) iterations. If not, then the processagain returns to step 212 at which another time delay is assumed, andfurther proceeds to repeat steps 208 and 210. However, if upon reachingstep 212 it is determined that there have already been N zero resultscorresponding to N different time delay values, then the process divertsto a step 214 at which a sum of all of the different resultscorresponding to the different assumed time delay values is calculated,and subsequently to a step 216 at which the process ends. The step 214is optional and, in some embodiments is not performed such that theprocess diverts directly to step 216 from step 210.

Due to the use of the assumed values of the time delay τ, thecross-correlation computation process of the flow chart 200 can beadvantageous in comparison with the previously-describedcross-correlation processes. In particular, by assuming the values ofthe time delay, computational effort need not be expended in determiningthe actual time delay value. Further, although numerous (e.g., fifty ormore) iterations need to be performed in some circumstances to reach thecriterion of the step 210 at which the iterations are stopped, this doesnot take excessive time since, given an appropriate digital signalprocessing (DSP) chip/device that performs time-shifting (e.g., any ofseveral DSP chips available from Texas Instruments, Inc. of Dallas, Tex.that are capable of performing several hundred million instructions persection (MIPS)), the different iterative calculations can be performedsimultaneously or nearly simultaneously in parallel. The speed at whichthe calculations are made in particular can surpass the flow rate of theparticles/beads within the fluidic channel.

It should also be noted that the result obtained from applying thecross-correlation algorithm (e.g., Equation (3)) during each iterationat the step 208 is representative of the amount or number of particlesflying by the waveguides of the waveguide array at a given speedcorresponding to the assumed time delay, and thus the individual resultsare of individual interest as being representative of the amount ofparticles passing through the fluidic channel at different speeds. Thiscan be valuable in the context of flow cytometry, particularly where itmay be expected (or of interest) that different cells or different DNAbase pairs travel at different speeds. At the same time, the sum of allof the individual results calculated optionally at the step 214 also canbe of interest, as an indication of the total sample intensity.

Referring additionally to FIGS. 11( a) and (b), these FIGS. provide anillustration of the effectiveness of the above-describedcross-calculation techniques when implemented in relation to the system80 of FIG. 6 with its FPIC device 100 having the array 54 of eightwaveguides. More particularly, FIGS. 11( a) and (b) respectivelyillustrate exemplary cross-correlation data that can be obtained byapplying Equation (3) to the raw data of FIGS. 8( b) and 8(c),respectively, with each of the FIGS. 11( a)-(b) displaying R(τ) as afunction of τ. Clearly the signals have been completely restored asmanifested by pronounced peaks 195 of R(τ) shown in each case. As shown,the maximum of R(τ) occurs at 0.3 sec and 0.08 sec for the two casesinvolving 5 μm and 1 μm beads, respectively. These are the durationsrequired for the respective particles to travel across two neighboringwaveguide channels of the array of waveguides. For a center-to-centerchannel spacing of 100 μm, the velocities of particles in each case are333 μm/sec and 1250 μm/sec respectively. Therefore, the eight-channelwaveguide array shows its superb ability of signal enhancement to allowfor time-of-flight measurement on even extremely weak fluorescent beads.The velocity obtained in this way is a direct measurement of particlespeed and can be used for in-situ calibration of the fluidic system.

Turning to FIGS. 12( a)-(b), for particle detection and sorting, it isfurther desirable to measure signals in real time as an intensity signalchain. To generate the signals shown in FIGS. 12( a)-(b), thenoise-masked raw data in FIGS. 7( a)-(b) obtained using the output ofthe 8×1 demultiplexer 76 and pertaining to the 5 μm and 1 μm bead data,respectively, is processed through Equation (1), with the value of Tobtained from the previous time-of-flight measurement. Distinctive peaks(e.g., groups of spikes) 180 with side-lobes 181 represent passing beadsin real time. From the results in FIGS. 12( a)-(b), it is evident thatthe values of the peaks 180 of the 5 μm beads are many (e.g., 17) ordersof magnitude greater than those of 1 μm beads, which is due to the factthat Equation (1) is a product of eight correlated signals and thereforesignificantly magnifies the difference in signal intensity. Theseresults suggest that the scheme of multi-channel detection not onlyimproves the signal-to-noise ratio but also enhances the ability todistinguish signals of slightly different intensity, which is animportant capability in the context of flow cytometry. (It should benoted that, in FIGS. 12( a)-(b), intensity can be of any arbitrary unit,and the dimension “number of frame” corresponds to time with, moreparticularly, one frame corresponding to 1/30 second.)

It should further be noted that, for detection and sorting in someconventional flow cytometry systems, emission intensity is used toidentify targets of different characteristics. Although cell sorting byintensity is the predominant and simplest method, it is not as reliableand accurate as desired when the intensity difference between normal andtargeted samples is small. As shown in FIGS. 12( a)-(b), the peaksproduced by passing beads show considerable differences in theirmagnitude even though these beads belong to the same group within avariation of a few percents in their size and shape. Variations in thevalues of the peaks 180 can be partly attributed to non-uniformity ofthe beads. These large intensity variations of these “similar” beadssuggest the ability of detection schemes in accordance with at leastsome embodiments of the present invention to distinguish samples of onlysmall difference, thus making cell detection/sorting by intensity morereliable and accurate.

In at least some further embodiments, it is desirable that compensationbe provided to account for variation in the distance of a given beadpassing each waveguide, which otherwise can cause signal intensitychange due to the variation of light coupling efficiency. In some suchembodiments, optical detection from the opposite side of the fluidicchannel can be utilized to eliminate the effects of positionalvariation, since any positional variation of a bead is supposed toproduce an anti-correlation signal between the oppositely-locatedwaveguides. For example, using a FPIC device similar to the FPIC device40 having the arrays 52 and 54, signals provided by the waveguides ofthe array 52 would compensate for the signals provided by the waveguidesof the array 54. If a bead is away from the center of the fluidicchannel, then the signal intensity from the waveguides near the bead(for example, the waveguides of the array 52) increases and the signalintensity from the waveguides farther from the bead (for example, thewaveguides of the array 54) decreases. This yields a negativecorrelation between the output signals from the waveguides on both sidesof the channel. At the same time, intrinsic property variations of thebead will produce a positive signal correlation. That is, when a beadhas a low fluorescence efficiency, output signals from the waveguides onboth sides are reduced. In addition, it should also be mentioned thatimproving the flow channel design such as using multiple stream laminarflows can also suppress the undesirable effects of positional variation.

Another noticeable feature for the signal peaks 180 in FIGS. 12( a)-(b)is the occurrence of the side-lobes 181. In an ideal case when thebackground noise is small, high main-lobe-to-side-lobe ratios should beobtained due to the algorithm of Equation (1). However, when the signalof each waveguide channel is comparable to the noise, the nonzerobackground potentially raises the magnitude of the side-lobes, which canbecome problematic particularly when the bead population increasessufficiently that the signals produced by neighboring beads interferewith each other through their side lobes. Nevertheless, it should benoted that the occurrence of side lobes or multiple peaks for a singlepassing bead is the result of using the simplest algorithm, namely, thatof Equation (1). If Equation (3) is instead employed such that a slidingtime scale is used to define the lower and upper limit of integrationtime interval, there will be no side lobes and the signal appears to bea sequence of peaks similar to those in FIGS. 11( a)-(b) in time domain.In that situation, the specific time for each peak represents thearrival time of the bead to the first waveguide. To avoid crosstalk, theflow rate should be controlled so that two beads are not passing thearray at any given time (so as to set a limit of the device throughputfor each fluidic channel, as well as possibly a system throughput, wherethe system throughput is the product of the throughput of each fluidicchannel and the total number of channels).

In addition to the above-described embodiments, the present invention isintended to encompass a variety of other devices, systems andtechniques/methodologies that include one or more of the above devices,systems and techniques/methodologies and/or portions thereof. Forexample, although not shown in the FIGS., in at least some additionalembodiments, a CCD connected microscope can be placed on top of thesample device to simultaneously monitor the events happening in thefluidic channel in order to verify the counting accuracy. By comparingthe monitoring video with the waveguide channel data obtained during thesame period of time, it is possible to address the correspondencebetween individual beads and peaks.

Although such a methodology involving both the waveguide signals andmonitoring video can be helpful, it is limited in two respects. First,the error rate determined by this methodology is only valid when theflow rate is slow, due to the restriction of the speed of the monitoringCCD. Further, the verified period of time can be limited, for example,because the image acquisition and process are executed by a personalcomputer. One way of achieving valuable data notwithstanding theserestrictions is to incorporate real-time signal processing and sortingfunctions with the current detection architecture, so that targetedparticles can be collected for counting and calibration. For example, toapproach such a solution, it is possible to design and utilize signalprocessing circuits that will trigger the sorting mechanism in real-timewhen events are detected. Also, for the sorting part, a new sortingmechanism using acoustic waves can also be introduced. It is intendedthat, in at least some embodiments, a flow cytometer with completefunctions of detection, signal processing and sorting will be integratedin a single chip platform.

Although not limited to applications relating to flow cytometry, atleast some embodiments of the present invention can offer significantcost, size, and performance advantages that have a potential to improveor even revolutionize conventional flow cytometry techniques. Thetechnology and the architecture design of FPICs in accordance with atleast some embodiments of the present invention significantly enhancethe detection sensitivity through multi-point detections, hence openingup the possibility of using low cost light sources (e.g., light-emittingdiodes (LEDs) and lamps) and detectors (e.g., semiconductor avalanchephotodiode detectors (APDs)) to replace mainframe lasers,photomultiplier tubes (PMTs), and lock-in amplifiers. It also offers newfunctions such as measurements of particle velocity, quantum efficiencyfluctuation, signal difference between similar samples, etc. that couldprovide new insights in relation to biosensing. Additionally, the FPICplatform offers a natural path to form array structures for parallelprocessing, which makes up for the possible throughput reduction due tothe lower flow rate of microfluidic circuits. As described above, FPICdevices can be made of polymer materials by way of simple yetcontrollable methods (e.g., micro-molding and capillary channelfilling), and the devices can be readily transferred to semiconductor orsilica substrates for integration with optoelectronic or electronicdevices.

As discussed above, a significant purpose of at least some embodimentsof the FPIC devices of the present invention when implemented foron-chip flow cytometry applications is to enhance the sensitivity offluorescent detection using the architecture of array waveguides thatprovides multiple detection zones for objects traveling through thefluidic channel. In addition, the waveguide arrays can performtime-of-flight measurement and multi-label fluorescent detection withwavelength filters. Further, the monolithic integration of waveguideswith microfluidic channels as described above is only one example of avariety of possible implementations of various structures on photonicICs to realize flow-cytometry-on-a-chip and for other purposes. Forexample, fabrication methodologies such as those described above (orsimilar to those described above) can be employed to incorporate morefunctional optical waveguide devices, such asmultiplexers/demultiplexers, power splitters, filters, polarizers, etc.to further enhance and expand the detection and analysis functions orfor other purposes.

Although both the eight detector and single detector designs describedabove produce superior sensitivity in comparison with conventionaloptical detectors, designs such as that of FIG. 4 having an integrateddemultiplexer are preferred in at least some circumstances, since insuch designs only one detector rather than multiple detectors in theform of a detector array is required. Also, in embodiments where asemiconductor APD or a PMT is used, the detector bandwidth (e.g., >1MHz) is more than sufficient to support the sampling rate (e.g., eighttimes of the particle throughput) for unequivocal detection of eachpassing particle, cell, bead, analyte, etc. In at least someembodiments, the use of an eight-channel waveguide array such as thatmentioned above also allows for other measurements to be made that canyield useful information. These include, for example, time-of-flightmeasurements and timing jitter measurements to monitor Brownian motionand flow effects. Such information makes it possible to track thebehavior of each individual particle in the fluidic channel, producinginsight into the particle properties and signals for downstream control.

As mentioned above, the present invention is not limited to applicationsrelating to flow cytometry but rather is intended to encompass a varietyof embodiments of devices, systems and processes that can be utilized ina variety of biomedical, biochemical, and other sensing applications.Also, while in at least some embodiments of the present invention, oneor more arrays of eight detection waveguides are arranged along one ormore sides of a fluidic channel, in alternate embodiments, lesser orgreater numbers of waveguides than eight waveguides can be employed(indeed, in at least some embodiments, only one detection waveguide ispositioned along one or both sides of the fluidic channel). Thewaveguides can be oriented in a perpendicular manner relative to thefluidic channel as discussed above, but in alternate embodiments can beoriented in any particular manner relative to the fluidic channel. Also,while the FPIC devices shown in FIGS. 3( a)-(b), 4 and 6 employwaveguide arrays arranged on one or two opposing sides of the verticalsections 48, 68 of the fluidic channels 50, 70, in alternate embodimentsit would further be possible employ two sets of waveguides that extendedat right angles relative to one another (with the vertical sectionsserving as the vertex), or at other angles relative to one anotherrather than only at 180 degree angles relative to one another. Indeed,in further alternate embodiments, three, four or possibly even morearrays of waveguides can be positioned extending away from the verticalsections along three or more planes.

Additionally, while the detection waveguides can be square orrectangular in cross-section as shown, in alternate embodiments thewaveguides can take on alternative cross-sectional shapes (e.g.,circular cross-sections). Although the sections 48, 68 of the waveguidescan be vertical as shown in FIGS. 4( a)-(b) and 5, the sections need notbe vertical but rather can be horizontal or oriented in another mannerand, in certain embodiments, can also be curved. Further, although theexcitation waveguides 42, 44, 62 and 64 shown in FIGS. 3( a)-(b) and 4are aligned with the vertical sections 48, 68, respectively of thefluidic channels, in alternate embodiments, any of a variety of othertypes of light sources (including simply light bulb(s)) could beutilized to illuminate the fluid flowing within the channels. In suchother embodiments, it would not be necessary that the light be shinedthrough/along the lengths of the fluidic channels as shown in FIGS. 3(a)-(b) and 4; indeed, in certain embodiments, the light can be directedtoward the fluidic channels from any direction. Indeed, the presentinvention is intended to encompass embodiments of FPIC devices in whichlight (or other electromagnetic radiation) is detected via one or morewaveguides or other conductive structures, but in which excitation light(or other electromagnetic radiation) is delivered to the fluidic channelby way of any of a variety of structures (including simply light bulbs),not merely by way of one or more waveguides.

Additionally, while the fluidic channels (e.g., the channels 50,70) ofthe devices described above are microfluidic channels, the presentinvention is further intended to encompass other embodiments of devicesand systems that employ combinations of fluid channels and waveguidesand/or electrodes even where the fluid channels are not “microfluidicchannels”. For example, the present invention is intended to encompassdevices having fluid channels having dimensions substantially greaterthan those considered as being “microfluidic” channels, e.g., channelshaving cross-sectional dimensions of greater than micrometers ormillimeters. Further, it is intended that the present inventionencompass methods of constructing FPIC devices that involve conventionaltechniques for manufacturing microfluidic channels, and then supplementthose conventional techniques with additional steps to integratephotonic components (e.g., waveguides) with those channels/carriers.

It is specifically intended that the present invention not be limited tothe embodiments and illustrations contained herein, but include modifiedforms of those embodiments including portions of the embodiments andcombinations of elements of different embodiments as come within thescope of the following claims.

What is claimed is:
 1. A device, comprising: a processor; and a memorycomprising processor executable code, the processor executable code whenexecuted by the processor configures the device to: receive at least onesignal produced by at least one detection device, the signalcorresponding to an interaction between scattered or fluorescent lightand at least one particle suspended in a fluid within a fluidic channel,the scattered or fluorescent light having been produced uponillumination of the fluid and the at least one particle as the at leastone particle flows through the fluidic channel from first positionwithin the fluidic channel to a second position within the fluidicchannel; perform a calculation based upon the at least one signal andone or more transit times required for the at least one particle toproceed between the first and the second positions within the fluidicchannel, wherein the at least one signal includes a plurality ofsignals, and wherein the calculation comprises determining a product ofa plurality of values corresponding respectively to the plurality oftime-shifted signals; and produce information indicative of at least onecharacteristic of the at least one particle.
 2. The device of claim 1,wherein the plurality of time-shifted signals are shifted relative toone another by the one or more transit times.
 3. The device of claim 2,wherein the one or more transit times are obtained prior to obtainingthe at least one signal.
 4. The device of claim 1, wherein the at leastone particle has a diameter of approximately 1 micrometer.
 5. The deviceof claim 1, wherein the at least one signal comprises two or moresignals that are received from two or more detection devices configuredto detect the scattered or fluorescent light when the at least oneparticle is at two or more positions within the fluidic channel.
 6. Adevice, comprising: a processor; and a memory comprising processorexecutable code, the processor executable code when executed by theprocessor configures the device to: receive at least one signal producedby at least one detection device, the signal corresponding to aninteraction between scattered or fluorescent light and at least oneparticle suspended in a fluid within a fluidic channel, the scattered orfluorescent light having been produced upon illumination of the fluidand the at least one particle as the at least one particle flows throughthe fluidic channel from first position within the fluidic channel to asecond position within the fluidic channel; perform a calculation basedupon the at least one signal and one or more transit times required forthe at least one particle to proceed between the first and the secondpositions within the fluidic channel, wherein the calculation comprisesiteratively performing two or more calculations based upon the at leastone signal and the one or more transit times, wherein each additionaliteration of the calculation results in a respective additional piece ofinformation.
 7. The device of claim 6, wherein the processor executablecode when executed by the processor further configures the device todetermine whether a threshold number of successive ones of therespective additional pieces of information have been determined to besubstantially equal to zero and, if so, cease to perform the additionaliterations.
 8. The device of claim 6, wherein the processor executablecode when executed by the processor further configures the device tocalculate a sum of the information and the additional pieces ofinformation, and output at least one of: the information, the sum, andderivative information based upon at least one of the information andthe sum.
 9. A device, comprising: a processor; and a memory comprisingprocessor executable code, the processor executable code when executedby the processor configures the device to: receive at least one signalproduced by at least one detection device, the signal corresponding toan interaction between scattered or fluorescent light and at least oneparticle suspended in a fluid within a fluidic channel, the scattered orfluorescent light having been produced upon illumination of the fluidand the at least one particle as the at least one particle flows throughthe fluidic channel from first position within the fluidic channel to asecond position within the fluidic channel; perform a calculation basedupon the at least one signal and one or more transit times required forthe at least one particle to proceed between the first and the secondpositions within the fluidic channel, wherein the calculation comprisescomputing a product between a time-varying intensity function of the atleast one signal and a time shifted version of the time-varyingintensity function of the at least one signal.
 10. The device of claim9, wherein the one or more transit times are obtained prior to obtainingthe at least one signal.
 11. The device of claim 9, wherein the at leastone particle has a diameter of approximately 1 micrometer.
 12. A device,comprising: a processor; and a memory comprising processor executablecode, the processor executable code when executed by the processorconfigures the device to: receive at least one signal produced by atleast one detection device, the signal corresponding to an interactionbetween scattered or fluorescent light and at least one particlesuspended in a fluid within a fluidic channel, the scattered orfluorescent light having been produced upon illumination of the fluidand the at least one particle as the at least one particle flows throughthe fluidic channel from first position within the fluidic channel to asecond position within the fluidic channel; perform a calculation basedupon the at least one signal and one or more transit times required forthe at least one particle to proceed between the first and the secondpositions within the fluidic channel, wherein the processor executablecode when executed by the processor further configures the device toperform the calculation by at least: (a) assuming a time value for eachof the one or more transit times; and (b) performing a cross-correlationcomputation on the at least one signal based on the assumed timevalue(s); (c) upon determination that the cross-correlation computationresult is not below a threshold value, assuming a new time value for atleast one of the one or more transit times and performing thecross-correlation computation based on the new assumed transit timevalue(s); and (d) upon determination that the cross-correlationcomputation result is below the threshold value, obtaining theinformation.
 13. The device of claim 12, wherein steps (a) through (c)are carried out iteratively for up to a maximum number of times.
 14. Thedevice of claim 12, wherein step (d) further comprises: producing acount as to the number of times the cross-correlation computation resulthas remained below the threshold value; and obtaining the information ifthe count is greater than or equal to a predetermined count, obtainingthe information.
 15. The device of claim 12, wherein step (d) furthercomprises computing a sum of all cross-correlation results obtained insteps (c) and (d).
 16. The device of claim 12, wherein thecross-correlation computation result is representative of number ofparticles that are suspended within the fluid as the fluid and particlessuspended therein flow through the fluidic channel.
 17. The device ofclaim 12, wherein the processor executable code when executed by theprocessor further configures the device to control or monitor anoperation of the at least one detection device.
 18. The device of claim12, wherein the threshold value is zero.